Lateral parabrachial FoxP2 neurons regulate respiratory responses to hypercapnia

About half of the neurons in the parabrachial nucleus (PB) that are activated by CO2 are located in the external lateral (el) subnucleus, express calcitonin gene-related peptide (CGRP), and cause forebrain arousal. We report here, in male mice, that most of the remaining CO2-responsive neurons in the adjacent central lateral (PBcl) and Kölliker-Fuse (KF) PB subnuclei express the transcription factor FoxP2 and many of these neurons project to respiratory sites in the medulla. PBclFoxP2 neurons show increased intracellular calcium during wakefulness and REM sleep and in response to elevated CO2 during NREM sleep. Photo-activation of the PBclFoxP2 neurons increases respiration, whereas either photo-inhibition of PBclFoxP2 or genetic deletion of PB/KFFoxP2 neurons reduces the respiratory response to CO2 stimulation without preventing awakening. Thus, augmenting the PBcl/KFFoxP2 response to CO2 in patients with sleep apnea in combination with inhibition of the PBelCGRP neurons may avoid hypoventilation and minimize EEG arousals.


Introduction
Patients with obstructive sleep apnea (OSA) have recurring arousals over the course of a night with loss of the upper airway muscle tone that results in obstruction of the airway, causing a reduction (hypopnea) or cessation (apnea) of ventilation, despite persisting respiratory efforts. The interruption of ventilation causes progressive hypercapnia and hypoxia, which in turn causes increased ventilatory effort. This increased ventilatory effort is measured by increased activity (as measured by EMG) of muscles related to ventilation, including both airway dilators such as the genioglossus (GG) and pump muscles such as the diaphragm, [1][2][3][4][5][6] and is associated ultimately with cortical arousal that further augments respiratory efforts that open up the airway and re-establish ventilation 5,[7][8][9] . However, these repeated awakenings cause sleep fragmentation, which is associated with deleterious cognitive, metabolic and cardiovascular consequences 5,7,8,[10][11][12][13][14][15] . The CO2 blood levels and ventilation increase rapidly during an apnea, but the fall in blood oxygenation typically lags behind the changes in CO2 blood levels and correlates poorly with the onset of the arousal 16-18 . We have therefore developed a mouse model of the repeated periods of exposure to elevated CO2 and brief arousal that mimics the events in OSA [19][20][21] . This model permits selective genetic targeting and manipulation of brain circuits that mediate the respiratory and EEG responses during the period of CO2 elevation associated with an apnea.
Our earlier work supports the hypothesis that the brain circuits that respond to hypercapnia by elevating respiratory efforts may be distinct from those that cause cortical arousal 19,20,22 . Speci cally, we found that glutamatergic neurons (i.e., expressing Vglut2) in the lateral PB complex show cFos activation during elevated CO2, and that deletion of Vglut2 from these neurons in the lateral PB (LPB) caused both a delay in arousal and reduced ventilation to a CO2 stimulus 20 . However, the two aspects of CO2 responses were not correlated across our deletions, which varied in their precise location, suggesting that the ventilatory and arousal responses were due to separate populations of glutamatergic neurons in the lateral PB region 20 . We later found that a subset of these neurons in the external lateral parabrachial nucleus that express the peptide CGRP (PBel CGRP neurons) play a critical role in transmitting the arousal signal to cause EEG desynchronization in response to hypercapnia, with minimal effect on respiration 19 . In addition, we observed a second population of non-CGRP neurons adjacent to the PBel CGRP group in the central lateral, lateral crescent and Kölliker-Fuse parabrachial subnuclei that are activated in response to CO2 exposure in mice [23][24][25][26][27][28][29][30][31] . Recent studies showed that neurons in the lateral PB expressing the transcription factor Fork head Box protein 2 (FoxP2) [32][33][34] have a distribution that is complementary to the PBel CGRP neurons including the regions containing the non-CGRP neurons that express cFos after CO2 exposure and which project to premotor and motor neurons that innervate the diaphragm and genioglossus muscles 35 . Therefore, we hypothesized that whereas the PB CGRP neurons wake up the forebrain during apneas, the adjacent PB FoxP2 neurons may trigger activation of ventilatory effort during apnea.
To test this hypothesis, we examined the role of the PB FoxP2 neurons in respiration and arousal response to CO2. We started by investigating their response (cFos expression) to CO2 exposure as well as in vivo recording of PB Foxp2 neuronal activity by using intracellular calcium imaging during spontaneous sleepwake and during exposure to CO2. We also investigated their contribution to the respiratory changes by optogenetically photo-activating PB FoxP2 neurons at different frequencies during either normocapnia or hypercapnia and by optogenetically photo-inhibiting them using archaerhodopsin T (ArchT), while mice were exposed to a CO2 stimulus.

Results
Activation of the PB FoxP2 neurons by CO2: cFos expression in the PB FoxP2 neurons: We subjected mice to either 2h of 10% CO2 (10%, CO2, 21% O2, 69% N2; n = 5) or normocapnic air (room air: 21% O2, 79% N2; n = 3) and then examined brain sections using immunohistochemistry for both FoxP2 and cFos, an immediate early gene, used as a functional marker for activity in neurons. Because both cFos and FoxP2 are transcription factors, they are localized to the nucleus of neurons (Fig. 1). Exposure to CO2 caused a large increase in number of FoxP2 neurons that also showed cFos expression in the KF (38.3 ± 5%) compared to room-air (8 ± 1%; F 1,7 = 21.88, P = 0.003; Fig. a1-d1 and f). The doubly labeled KF neurons formed a cluster located medial to the ventral spinocerebellar tract along the ventrolateral surface of the rostral PB. Slightly more caudally in the lateral PB, large numbers of neurons that showed cFos activation in response to CO2 exposure were found in the PBel (non-FoxP2 neurons, in the region of the CGRP neurons) and in the central lateral PB, just dorsal to the CGRP cluster and wrapping around its lateral margin. 49 ± 5% of the total cFos immunoreactive neurons in the PBL were FoxP2 neurons (F 1,7 = 11.95, P = 0.013; Fig. a2-d3 and f). After CO2 exposure, the majority of the cFos + neurons in both the PBcl (78 ± 2%) and in the KF (71 ± 4%) showed FoxP2 expression (PB-F 1,7 = 31.85, P = 0.001; KF-F 1,7 = 9.8, P = 0.02) compared to the 42 ± 8% in PBcl and 52 ± 4% in KF of the room-air group (Fig. 1e). Of the total population of FoxP2-expressing neurons in the PBcl (PB FoxP2 neurons) 30 ± 4% expressed cFos after CO2 exposure compared to 9 ± 3% in the control mice (room-air). Therefore, the PB FoxP2 and KF FoxP2 neurons constitute the bulk of the non-CGRP neurons in the region adjacent to the PBel CGRP neurons that were also responsive to CO2. Interestingly, we found a small number of KF FoxP2 neurons that express the gene for CGRP (Calca) (supplementary Fig. 1), by using orescence in situ hybridization for CGRP in FoxP2-L10 mice. The FoxP2 + CGRP neurons in the KF (mean diameter 18 ± 0.7 µm) tend to be signi cantly smaller (F 1, 18 = 72.4; P < 0.001) than the ones that are not FoxP2 + (26.3 ± 0.7 µm).
In vivo measurement of PB FoxP2 neuronal activity by ber photometry: To capture and analyze the pattern of activation of the PB FoxP2 neurons by CO2 in vivo, we injected an adeno-associated viral vector (AAV) expressing Cre-dependent GCaMP6s into the lateral PB of Foxp2 tm1.1(cre)Rpa /J (FoxP2-Cre) transgenic mice (developed by Dr. Richard Palmiter at University of Washington, and donated to the Jackson Laboratory) 34,36 . We double-labeled sections through the PB from FoxP2-Cre::L10 reporter mice (in which FoxP2 cell bodies are marked by GFP) with FoxP2 immunohistochemistry (which stained nuclei of FoxP2 expressing neurons magenta) to verify that Cre-recombinase was expressed eutopically in FoxP2 expressing neurons (supplementary Fig. 2a-d). All of the neurons transfected by an AAV that expressed Cre-dependent GCaMP6s (which also uoresces green) also showed Foxp2 expression in their nuclei (red in Fig. 2b). The implanted glass ber targeting the GCaMP expressing PB FoxP2 neurons allowed us to record intracellular calcium levels, a surrogate for neuronal activity (Fig. 2c). The population activity of the PB FoxP2 neurons was then correlated to the identi ed sleep-wake states, respiration and their changes in response to repetitive CO2 exposures.
In n = 5 mice, optical bers were located in proximity to the GCaMP expressing PB FoxP2 neurons (Fig. 2b).
A representative example of a ber photometry recording across an exposure to CO2 is illustrated in Fig. 2d. In these mice, GCaMP uorescence increased when the mouse transitioned from NREM sleep to either wake or REM sleep state (supplementary Fig. 3a and b). The increased activity of PB FoxP2 neurons when emerging from NREM sleep may contribute to the sudden increase in respiratory effort when an animal exposed to CO2 awakens from NREM sleep.
All trials with 30 sec exposures to 10% CO2 also resulted in cortical arousal in the latter half of the trial. Therefore, to avoid the confounding effects of signi cantly higher GCaMP responses caused by transition to wakefulness, we chose to analyze only the rst 15s of the trial segments, which in all cases preceded cortical arousal. Although the actual level of uorescence during exposure to CO2 waxed and waned during individual exposures (see heatmaps in Fig. 2e), when the level of ΔF/F (change in GCaMP activity/ background uorescence) was summated across trials (Fig. 2f) and over 15s prior to and during CO2 in all trials, there was a robust and statistically signi cant increase in uorescence of FoxP2 neurons during exposure to CO2 (F 1, 1682 = 62.2; P < 0.001) (Fig. 2f, ΔF/F normalized to pre-CO2), with the uorescence peak for each of the 59 trials during CO2 exposure increasing by 6.4 ± 0.9% (F 1, 116 = 45.95 P < 0.001) compared to pre-CO2. This increase in ΔF/F was also accompanied by an increase in respiratory efforts in response to CO2 as measured in 59 trials from 5 mice (Fig. 2g-i). During this period we found a signi cant increase in respiratory rate (RR, 40 ± 1% increase for the last 5 breaths of the 15 sec interval after onset of CO2 exposure compared to the last 5 breaths prior to onset of CO2; F 1, 1662 = 5.7, P < 0.001, Fig. 2h), and a smaller increase in tidal volume (V T ) that lagged behind the increase in RR (Fig. 2 h-i) but was statistically signi cant (28 ± 0.89% increase) for the last three breaths of the 15 sec interval (P = 0.025; P = 0.004 and P = 0.037) (Fig. 2i). The GCaMP uorescence (ΔF/F) is also shown as heat-maps for all the trials for 15 s before and after CO2 exposure (Fig. 2e). Analysis of this data shows that the average latency of the increase in intracellular calcium (Ca i ) to its rst peak was 9.6 ± 0.57 sec, and in only 13.5% of the trials was the latency less than 5 sec (Fig. 2f). This observation aligns with the rise in RR starting at about 5 sec (approximate time that it takes for CO2 levels to rise in the chamber and then reach the circulation). The RR increased progressively in all trials for at least the next 10 sec (Fig. 2h), and then increased abruptly at the time of cortical arousal, which occurred between 15 and 30 sec in all trials.
We then analyzed the neuronal calcium activity of 28 neurons for the periods where the mice (n = 3) were exposed to CO2 (Fig. 3b and c) in a plethysmograph that allowed us simultaneously to record their breathing as well as EEG and EMG. For this analysis, we wanted to prevent confounds introduced by cortical arousal (EEG desynchronization and increase in motor activity), so we reduced the CO2 concentration to 8% and only used trials that showed no cortical arousal during 30s of CO2 exposure and in the subsequent 15 sec when CO2 levels in the plethysmograph were still high (see Fig. 3d, also video-CO2 responsive neurons.mp4). In these experiments 8% CO2, caused a 65 ± 4.4% increase in RR (F 1, 530 = 4.95, P < 0.001, pre vs. post) to 249 ± 5 bpm 20-40sec after CO2, compared to 152 ± 2 bpm at pre-CO2 levels. Minute ventilation also increased signi cantly (MV, F 1, 530 = 5.8, P < 0.001) with a 87.5 ± 4.4% increase 20-40 sec after CO2 onset (22.3 ± 0.4 ml/min) compared to pre-CO2 levels of 12 ± 0.26 ml/min (Fig. 3d, f). V T showed a smaller but still signi cant increase (F 1, 360 = 1.53; P = 0.043) by 28 ± 5% during 20-40sec after CO2 onset (0.094 ± 0.002ml) compared to 0.074 ± 0.0016ml at pre-CO2. An example of simultaneous neuronal activity pro les for 9 cells during one representative CO2 trial is shown in Fig. 3d. We analyzed the activity of all n = 28 cells, for 30s before and for 90s after the CO2 stimulus in 4-7 trials during which the animals remained in NREM sleep for this period of time, and plotted the normalized uorescence (ΔF/F) as a heat map (Fig. 3e). A small number of cells (4/28, 14%) were more active prior to the CO2 exposure, and were less active during the exposure. We termed these CO2-Off cells. The remaining 24 CO2-On neurons all showed periodic increases in Ca i after exposure to CO2, with 17/24 cells showing three distinct uorescence peaks during and after CO2 exposure. 23/24 cells showed a rst peak in activity with a mean latency of 11 ± 0.6 sec after onset of the CO2, 21/24 showed a second peak at a mean of 27 ± 1.6 sec, and then 17/24 at 41 ± 2.8 sec; 1/24 only peaked at 33s after CO2 onset (Fig. 3f). Of the 17 cells that showed all three peaks, 10 cells peaked rst at 11 ± 0.21 sec after CO2 onset and then showed periodicity of 17-19 sec for appearance of the second and third peak. Smaller numbers of neurons (10)(11)(12) also showed peaks in activity at roughly 60 and 75 sec. None of these neurons displayed this pattern of periodic and synchronized activity during NREM sleep prior to CO2 exposure, although the periodic waves of activity were similar to those we observed in individual PB FoxP2 neurons in animals breathing room air during wake and REM sleep. These data suggest that PB FoxP2 neurons may have a tendency to be activated in periodic waves that are synchronous across the population during wake and REM, but that these waves of activity may be suppressed during NREM sleep. Exposure to CO2 may then remove this suppression, allowing resumption of periodic, synchrous waves of activity with a periodicity during CO2 exposure in NREM sleep of about 15 sec. Whether these synchronous waves of activity originate from a rhythmic pattern of input, or are generated by recurrent activity in a local network or even are a cell autonomous response is not clear.

Photo-activation of PB FoxP2 neurons and respiration:
To test the effect of activation of PB FoxP2 cells on respiration, we injected the PB of FoxP2-cre mice (n = 10) bilaterally with cre-dependent AAV-Flex-ChR2-mCherry (Fig. 4a), and implanted them for EEG/EMG recording along with bilateral optical bers to target illumination of the ChR2 expressing FoxP2 neurons in the PB (Fig. 4b). We recorded animals for sleep and breathing in normocapnic room air and during activation of ChR2 with a blue laser (473nm) using 10ms pulses at 5, 10 or 20Hz, for either 5s or 10s every 5 minutes. Trials in which mice were in NREM sleep for at least 30s before stimulation were analyzed for the effect of optostimulation on respiration. In 6 out of 10 implanted mice, the optical bers accurately targeted the ChR2 expressing PB FoxP2 neurons as shown in Fig. 4b. These animals consistently showed respiratory responses during optostimulation but also had EEG arousal either late in the stimulation or shortly after offset (Fig. 4c, d) in 70% of the trials. In the four cases where ber implants were placed either medial or lateral to the PB FoxP2 ChR2 expressing neurons, neither 10 nor 20Hz stimulation produced any effect on respiration or cortical arousals. To distinguish the increment in ventilation due to waking, we measured RR and V T for 5 breaths pre-stimulation, 5 breaths just before the end of stimulation (or just before onset of arousal, whichever occurred rst) and 5 breaths after the cortical arousal.
While breathing normocapnic air, 10Hz stimulation progressively increased RR (18.5% with 5 sec and 42% Stimulation at 20 Hz increased RR by 35% at 5 sec vs. 69% at 10 sec (F 1, 106 = 122.9, P < 0.001) and MV by 25% at 5 sec and 40% at 10 sec (Fig. 4f). This pattern was consistent with the ber photometry results, in which RR began to increase at the same time as calcium activity of PB FoxP2 neurons, but increases in V T lagged by about 10 sec. However, no signi cant changes on respiration were observed with photostimulations at 5 Hz (Fig. 4f).
To explore the interaction between optogenetic stimulation and activation of PB FoxP2 neurons due to elevated CO2, we repeated the 20Hz, 10 sec stimulation in mice breathing 2% CO2 (Fig. 4e, representative example). First, we observed that the presence of 2% CO2 throughout the experiments dramatically decreased the latency of awakening to the laser stimulus to 2.22 ± 0.3 sec compared to 21.7 ± 1.7 sec in normocapnic air (F 4, 27 = 3.2; P = 0.031) (Fig. 4d). Despite the relatively brief period of laser stimulation before awakening in 2% CO2, we also found a large and almost immediate increase in RR (81%; F 2, 12 = 44.25; P < 0.001) and MV (38%; F 2, 12 = 8.08; P = 0.006), with no change in V T (Fig. 4g), similar to that found with stimulation in normocapnic air (Fig. 4f). Interestingly, the EEG arousal occurred at around the same RR as with CO2 or optogenetic stimulation alone (i.e., approximately 240-250 bpm), raising the possibility that the arousal caused by stimulation of the PB FoxP2 neurons may be due to the sensory feedback from the increased ventilatory effort, rather than being a primary effect of the PB FoxP2 neurons.
Further, we used the mice from these experiments (n = 6) to map the projections of the ChR2-expressing PB FoxP2 neurons (Fig. 5a). We found extensive mCherry labeling of bers and terminals in the pre-Bötzinger complex (PBZ) and caudal ventrolateral medulla (CVL), and more moderate numbers of labeled terminals in the nucleus of the solitary tract (NTS) and hypoglossal nucleus (XIIn) ( Photo-inhibition of PB FoxP2 neurons during exposure to CO2: To test the necessity of PB FoxP2 neurons in producing the respiratory responses to CO2 during sleep, we photo-inhibited them during hypercapnia. FoxP2-cre mice were injected in the ventral part of the LPB with AAV-Flex-ArchT (n = 12) (Fig. 6a) and implanted with EEG/EMG electrodes and bilateral optical bers targeting the PB FoxP2 neurons ( Fig. 6c1-c5). We analyzed the respiratory responses to 10% CO2 given for 30s every 300s, with and without photo-inhibition of the PB FoxP2 neurons using a 593 nm laser light (Fig. 6b). The laser light was on for 60s, beginning 20s prior and extending to 10s after the CO2 stimulus (30s) (representative trials of CO2 exposure are shown in Fig. 6 d and e).

Discussion
Our previous work identi ed a population of CGRP neurons in the PBel that project to the forebrain and are required for arousal from sleep during CO2 exposure, but make little contribution to respiratory efforts.
Here we report a second adjacent population of neurons that is marked by expression of the FoxP2 transcription factor and is required for normal respiratory response to CO2 during sleep, but not arousal.
These PB FoxP2 neurons are located in clusters in the PBcl just dorsal to the CGRP group and in the KF subnucleus, and along a narrow bridge just lateral to the CGRP group (the lateral crescent) connecting the two major clusters. This population of PB FoxP2 neurons projects intensely to medullary respiratory areas as well as to hypothalamic targets (lateral hypothalamus, preoptic area) associated with regulation of wake-sleep. However, optogenetic stimulation of the PB FoxP2 neurons causes immediate increases in ventilation, but only delayed and inconsistent arousals, and while photoinhibition reduces the ventilatory increases caused by CO2 it does not affect arousal. Hence, we interpret the PB FoxP2 neurons as primarily mediating the ventilatory response to CO2.
We further found that optogenetically activating the PB FoxP2 neurons eventually also wakes the animal up, although often after the PB FoxP2 neurons are no longer being stimulated, and at levels of ventilatory effort that are similar to that at the time of arousal in an animal exposed to 10% CO2. We interpret these ndings as an indication that CO2 exposure largely and independently activates populations of PB CGRP neurons that cause EEG arousal and PB FoxP2 neurons that cause increased ventilation. However if animals are forced to increase ventilation enough, they are awakened by the effort, and if they wake up during CO2 exposure, they further increase ventilation. This relationship underscores the synergy between the behavioral and respiratory motor responses to insure survival when an animal is either apneic or asphyxiated.
We used three different intersecting strategies to identify the role of the PB FoxP2 in the ventilatory response to CO2 during sleep. First, we showed that among the neurons in the PBcl and KF (i.e., outside the PBel CGRP group) that show cFos expression after CO2 exposure, nearly 75% express FoxP2. This PB FoxP2 population had no overlap with the PBel CGRP neurons (i.e., no PBel CGRP neurons expressed FoxP2), indicating that they are independent cell populations. Interestingly, we found a small number of KF neurons that are CGRP-immunoreactive (supplementary Fig. 1). These neurons are a bit smaller than those in the PBel, and as shown by Geerling and colleagues 34 , they project to respiratory sites in the medulla, like the remainder of KF glutamatergic neurons (see also Yokota et al., 2015 35 ). We had missed this population in our previous reports because they appear to express CGRP at a lower level than the larger PBel CGRP neurons (Nardone, Saper and Lowell, in preparation) 37 . We use Calca-ires-Cre-ER mice in our experiments for identifying and manipulating CGRP neurons, and apparently the level of Cre expression in these small KF CGRP neurons was not su cient to cause recombination events. By contrast these neurons appear to have greater Cre expression in the Calca-Cre knockin mice used by Geerling et al., and so are more readily identi ed. Interestingly, our in situ hybridization results reported here (supplementary Fig. 1) indicate that at least some of the KF CGRP neurons also express FoxP2, underscoring that they are a separate population from the PBel CGRP neurons and demonstrating the value of FoxP2 as a marker for identifying the neurons that drive ventilatory responses to CO2. It should be pointed out, however, that FoxP2 is expressed more widely in more dorsally located neurons in the lateral PB, but only the FoxP2 neurons in the PBcl, KF and lateral crescent express cFos in response to CO2. We are currently searching for more selective markers for this neuronal population.
The second strategy we used to investigate the relationship of the PB FoxP2 neurons with the ventilatory response to CO2 was the employment of ber photometry to study the intracellular calcium responses of PB FoxP2 neurons during wake-sleep and during CO2 exposure. The PB FoxP2 neurons were clearly wake and REM active. This nding is consistent with the general reduction of respiration during NREM sleep, and the fact that arousal from NREM sleep caused a rapid increase in both ventilation and in the calcium signals from the PB FoxP2 neurons. This population of neurons showed a brisk increase in calcium signal beginning a few seconds after onset of a 10% CO2 stimulus, but persisting for many seconds after the stimulus was removed. We then resolved the responses of individual PB FoxP2 neurons with GCaMP6s endoscopy during exposure to 8% CO2 (a concentration chosen because it generally did not wake up the animals during a 30 sec exposure). Surprisingly, the individual PB FoxP2 neurons showed multiple recurrent peaks of calcium activation, with the rst peak at 11.0 ± 0.2 s after onset of the gas and then further peaks at roughly 17-19 sec intervals. The mechanism for these rhythmic pulses of activity in the PB FoxP2 neurons remains a mystery. These experiments admittedly involved only a small number of PB FoxP2 neurons and because of the limited depth of eld of the GRIN lens we used, all of them were in a small cluster near the surface of the lens. However, if the larger population of PB FoxP2 neurons also responds rhythmically to CO2, our results would suggest that the much smoother population response as imaged by ber photometry is probably a summation of the activity of different subsets of PB FoxP2 neurons that are activated at different times in different locations in the PBcl and KF during CO2 stimulation. Our anterograde and retrograde tracing experiments indicate that PB Foxp2 neurons may mediate these effects on respiration by their descending projections to the medullary targets involved in ventilatory rate and volume 35 .
The third strategy we used to characterize the role of the PB FoxP2 neurons in the response to CO2 involved optogenetic excitation and inhibition. When the PB FoxP2 neurons were opto-stimulated at 10 or 20Hz while mice were breathing normocapnic air, they showed a statistically signi cant increase in RR and MV (but not V T ) that was greater both with higher frequency or prolonged duration of stimulation.
Interestingly, the animals only awakened either late in the stimulation episode or immediately afterward, suggesting that the EEG arousal was not a direct effect of the stimulation. In this regard, it was interesting that when animals were stimulated optogenetically while breathing 2% CO2, the latency of the arousal was shorter, but occurred at about the same RR as the opto-stimulation while breathing room air or in response to CO2 without optostimulation (i.e., awakening in all three conditions occurred at about 240-250 breaths/min, or when RR was about 50% greater than baseline). This observation suggests that the arousal may have been due to somatomotor feedback from the increased ventilatory effort.
Alternatively  43 . These ndings suggest that the PB FoxP2 neurons may target the pre-Botzinger complex to cause the increase in RR (Fig. 7).
By contrast, photo-inhibition of the PB FoxP2 neurons during a 30 sec exposure to CO2, did not prevent the rise in RR but did diminish the rise in V T and MV, not only during the CO2 exposure but even after the cortical arousal, when the largest increase in V T is generally observed. On the surface it may appear that the increase mainly in RR with photostimulation of the PB FoxP2 neurons and decrease mainly in the V T with photoinhibition of the same cells is inconsistent. However, the photostimulation was done while the animals were breathing room air, so the increase in RR cause by the PB FoxP2 neurons was not being masked by the contributions of other brainstem sites (such as the retro-trapezoid nucleus, para-facial complex, medullary raphe and the nucleus of solitary tract) that are also engaged by high CO2 levels 41,43,[45][46][47] . In addition, although the increase in V T was not statistically signi cant during these brief periods of stimulation, V T changes with hypercarbia lag considerably behind the increase in RR. On the other hand, during 10% CO2 stimulation, when these other CO2-responsive sites were already increasing RR to its maximum, 44, 48-50 the inhibition of PB FoxP2 neurons revealed although they are not needed to reach maximal RR, they are necessary to achieve the maximal increase in ventilatory volume. In other words, brief activation of the PB FoxP2 neurons is su cient to drive RR (but not V T ) when breathing room air, and necessary to drive increased V T during exposure to high CO2 levels [51][52][53][54] . A schematic model describing the proposed neuronal circuit for hypercapnia induced increase in ventilation implicating the role of PB FoxP2 neurons is shown in Fig. 7.
In conclusion, our experiments reveal that PB FoxP2 neurons likely contribute both to the increases in RR and V T during exposure to CO2 in NREM sleep, but that they are not the only pathway driving these responses (Fig. 7). On the other hand, if ways could be found to augment the PB FoxP2 response to CO2, this may be su cient to avoid hypoventilation and minimize EEG arousals in patients with obstructive sleep apnea. However, activating the PB FoxP2 neurons to increase RR by 50% or more above baseline may by itself contribute to EEG arousal. It is not known whether this arousal is due to the mechanical sensory feedback of increased ventilatory effort, or might represent collaterals from the PB FoxP2 neurons that indirectly activate PBel CGRP neurons or some other arousal pathways. However, for activation of PB FoxP2 neurons to be effective in treating patients with obstructive sleep apnea, it will be important to nd ways to suppress the EEG arousal while augmenting the respiratory response to CO2.

Methods
Animals: We employed Foxp2 tm1.1(cre)Rpa /J transgenic mice in which IRES-Myc tag-nuclear localization signal (NLS)-cre-GFP-frt-neomycin-frt were introduced just after the termination codon of the mouse Foxp2 gene via homologous recombination. The mutation was created via homologous recombination in (129S6/SvEvTac x C57BL/6) F1-derived G4 embryonic stem (ES) cells. The frt-anked neomycin cassette was excised through crosses with animals that broadly expressing Flp recombinase. The GFP is believed to be nonfunctional. Resultant mice were backcrossed to C57BL/6J for 9 generations by the donating laboratory to the Jackson laboratory (Strain #:030541; RRID:IMSR_JAX:030541). All transgenic mice used here were heterozygous for the transgene and backcrossed to the C57BL6 strain and wildtype littermates were used as controls. We bred these mice in our animal facility and con rmed their genotype by using a Red Extract N-amp Tissue PCR kit (Sigma Aldrich; Catalog # XNAT-1000RXN) and Cre forward and reverse primers to detect the Cre recombinase gene. Their wildtype litter mates were used as controls, in each experiment.
Validation of mice: To test if the Cre expression was eutopic with FoxP2 expression FoxP2-Cre mice from Jackson Labs, we also validated by crossing them to the L10 reporter mice. The cre-positive neurons were labeled with green uorescent protein (GFP), and when the tissue was immuno-stained for FoxP2, we could observe nearly all green (GFP) neurons expressed labeling for Fox-P2 (red) in their nuclei (supplementary Fig.2a-d). This con rmed that we could reliably use these mice for expressing credependent virus vectors in the FoxP2 neurons in the PB, and then use ber-photometery or optogenetics to record their activity pro les or manipulate them (examples of cre dependent transfections- Fig.2b,4b and 6c). PB Foxp2 neurons did not overlap with the PB CGRP neurons in the lateral PB.
All mice used in these experiments were male because female mice of the same age are smaller, and including animals of various sizes would introduce noise into analysis of respiratory volumes (which scale with body size) across groups. Animals were maintained on a 12 h light/dark cycle with ad libitum access to water and food and were singly housed after surgery, with ambient temperature of 21-23 o C and humidity levels between 40-60%. Male littermates were randomly assigned to the experimental groups. All animal procedures met National Institutes of Health standards, as described in the Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee.
Vectors: For ber-photometry we used AAV-pSyn-GCaMP6s from Addgene_100843. For optogenetic experiments, we used the excitatory opsin (AAV-EF1a-DIO-hChR2(H134R)-mCherry-AAV-serotype8) and the optogenetic neural silencer AAV-CAG-FLEX-ArchT-GFP (AAV-serotype-8). These viral vectors was procured from the University of North Carolina (UNC) vector core and has been previously used for excitation as well as for silencing neurons and their terminals by us 19,55 . The viral vectors expressing excitatory and the inhibitory opsins were packaged at the UNC vector core.
Surgery: Under surgical anesthesia, mice were instrumented for sleep with implantation of EEG and EMG electrodes in addition to implanting either the ber photometry cannula (unilateral), or integrated grin lens with baseplate (unilateral) or bilateral optical bers targeted to the PB area (AP: -5.1 to -5.3mm; DV-2.6mm; ML: ±1.3mm). All implants were done after 5 weeks post injection of the injections of the viral vectors in the PB, to ensure optimal expression of the gene expression. After recovery from surgery, mice were recorded for sleep and respiration after acclimatizing them to the recording apparatus for at least once in a week before the actual recordings were performed. We recorded the arousal and respiratory responses to CO2 by placing them in the plethysmograph and recording them for both sleep and breathing by the procedure described previously 19,21,55 .
Experiment 1a: C57BL/6J mice (n=8) were moved from the animal housing room and placed in the plethysmographs where they were exposed continuously to either hypercapnic (21% O 2 ; 10% CO 2 ; 69% N 2 ) or room air (21% O 2 ; 0% CO 2 ; 79% N 2 ) for two hours, after rst habituating them to the chamber at room air for two hours. Air or hypercapnic exposures were performed between 10:00 and 12:00. At the end, mice were deeply anesthetized with chloral hydrate (500mg/kg, ip) and transcardially perfused with saline, followed by 10% formalin. Brains were removed, post-xed overnight immersed in 20% sucrose and cut into four alternate series of 30 micron frozen sections. After immune-staining the tissue for cFos and FoxP2, using speci c antibodies as per the methods described below, the cells that were double labeled for cFos and Foxp2 were counted in the PBcl (0.5mmX 0.6mm placed dorsal to the single labeled cFos in the PBel, as in Fig. 1a2-d3) and KF (0.5mm X 0.5 mm placed ventral to the ventral spino-cerebellar tract, as in Fig. 1a1-d1) sub-nuclei, after scanning the slides using a slide scanner (Olympus VS200 slide scanner) and acquiring the images in Olympus OlyVia 3.3 software using square grids. To avoid counting bias, the cell counts were performed by investigators blind to the treatment groups (NL and JDL). The original gures in Fig1a-d were taken by a confocal laser microscope (LasX Leica DMi8 confocal). We counted the sections that were separated by at least 120µm, and this also involved counting only the nuclei for both the FoxP2 and the Cfos, that reduced the chances of counting cells twice. Cell counts were also corrected for the cell size using the Abercrombie correction factor.
Experiment 1b: Fiber-photometry implants: In vivo calcium monitoring during sleep-wake and with CO2 exposures was performed using ber photometry. After 5 weeks of the viral injection of AAV-pSyn-GCaMP6s in the PB, laser light was passed into the brain via low-uorescence ber optic patch cord (0.48NA, Doric Lenses) connected to the implanted ber optic cannula with a metal sleeve (Doric Lenses, MFC_400/430-0.48_5mm_MF-1.25). Using the patch cord, we simultaneously delivered light via LED drivers at 465 nm and 405 nm (Doric Lenses, CA), to measure the calcium-dependent and calciumindependent (UV, isobestic), excitation of the GCaMP. Excitation emission from the GCaMP protein passed back through the ber optic patch cord and through the uorescence Mini Cube (Doric Lenses) and detected by a photo-receiver (Doric Lenses). Signals detected by the photo-receiver were transmitted to Axon Digidata 1322A analog-to-digital converter and the signals were acquired using Axoscope software-v10 (Molecular Devices, Foster City, CA, USA), alongside the EEM/EMG and breathing signals. We used exported les to Spike2 and analyzed the respiratory signals using the spike respiratory scripts (Resp80t, Spike2, CED, UK) that were then correlated with the GCaMP activity.
The GCaMP6 raw data was normalized to the baseline uorescence of each trial to obtain the ΔF/F (change in uorescence intensity relative to the baseline uorescence intensity) for different animals. The GCaMP ΔF/F values per second was calculated for 15 s before and during CO2 for each trial and statistically compared, along with RR and VT values for similar periods. The peak values of GCaMP ΔF/F, and the latency to peak during the entire 15s with CO2 exposure were also calculated. Two-way ANOVA was performed to compare the effects between pre and post CO2 exposures on the GCaMP ΔF/F and also for RR and VT.
Experiment 1c: GRIN lens implants: Foxp2 tm1.1(cre)Rpa /J mice (heterozygous FoxP2-Cre), after 3weeks of the virus injection (AAV-pSyn-GCaMP6s) in the PB were implanted with a microendoscopic ProView™ Integrated Lens 0.6mm x 7.3mm (Inscopix Catalogue #1050-004413) that allowed for visualizing the activity during the lens implant. The lens was targeted to be ~200-300 µm above the neurons using the following coordinates--5.0mm posterior to bregma, -1.4mm lateral from midline and -2.8 to 3.0 mm ventral to the dura mater. The baseplate provide the interface for attaching the miniature microscope during the calcium-imaging experiments, but during other times a baseplate cover (Inscopix catalogue # 100-000241) was attached to prevent damage to the micro endoscopic lens. Out of approximately 8 mice injected with GCaMP6s virus, 4 had successful implants and were used for the study.
Calcium imaging: We imaged the calcium activity at 5 frames per second, 200-ms exposure time at 20-30% LED power using the miniature microscope from Inscopix (nVista). These parameters caused minimal bleaching, and allowed long term recordings in mice. Mice were recorded for 5 min every hour for correlating the calcium activity to the 12h sleep-wake behavioral data. For correlating to the CO2 induced respiratory changes, mice were recorded in a plethysmograph, where they were subjected to repeated stimulus of 30s of 8% CO2 every 5 minutes, and the imaging was done for 4 trials (~20 min) every hour for 3h after 3-4h of habituation of mice to the recording chamber with every recording. This protocol caused minimum bleaching and allowed repetitive 2-3 recordings in an animal with a week of separation between subsequent recordings. neurons, we injected an adeno-associated viral vector expressing the excitatory opsin AAV-FLEX-ChR2-mCherry in the PB (AP: -5.1 to -5.3mm; DV-2.6mm; ML: ±1.3mm) and implanted these mice (n=10) with bilateral optical bers targeting the lateral PB. We also injected some wild type mice (n=3) with AAV-FLEX-mCherry as well, which served as control, we did not observe any expression of ChR2. We recorded these mice for sleep and respiration when they were exposed to the room air as per the method previously described. Mice were subjected to 5 or 10s of 467nm laser stimulus with pulse width of 10ms and with frequencies of either 5Hz or 10Hz or 20Hz, in a random order and with each treatment separated by at least 7-10 days. Histology: At the conclusion of the experiments, the animals were perfused with 0.9% saline followed by 10% buffered formalin while under deep anesthesia. Brains were harvested for analysis of the effective location of the injection site. Brains were kept in 20% sucrose for 2 days and sections were cut at 30mm using a freezing microtome in four 1:4 series.
Immunohistochemistry: C-Fos: Oncogene Sciences, cat # Ab5, rabbit polyclonal, raised against amino acids 4-17 of human c-Fos. This antibody stained a single band of 55 kDa on Western blots from rat brain (manufacturer's technical information). Sections were processed for detection of c-Fos alone or c-Fos in combination with FoxP2. All incubations were performed on free-oating tissue sections at room temperature. Sections were rst incubated overnight in c-Fos antibody. When c-Fos staining was to be combined with FoxP2, we used Rabbit anti-Fos antibody (diluted 1:10K in PBS with 0.2% Triton X-100).
Neither of these antibodies showed immunostaining when the primary antibodies were omitted, and when the tissue from control mice was used that were not injected with viral vector. Some of the brains (n=3) from WT mice were injected with CTb and were immunostained using Goat anti CTb ( Fluorescent In situ hybridization (FISH using RNA Scope): We identi ed FoxP2 neurons by using Foxp2 tm1.1(cre)Rpa /J mice crossed with R26-lox-STOPlox-L10-GFP reporter mice (FoxP2-L10, n=3), and labeled for Calca (the gene for CGRP) by using a set of FISH probes with RNAScope in brain sections from the KF and PBcl areas. The brain was sectioned at 30 µm and sections were mounted on glass slides in RNAs-free conditions, and RNA scope was performed using the multiplex uorescent reagent Kit V2 (Cat# 323100, Advanced Cell Diagnostics, Hayward, CA). Brain sections on the slides were pretreated with hydrogen peroxide for 20 min at room temperature and then with target retrieval reagent for 5 minutes (at temperature above 99°C), followed by dehydration in 90% alcohol and then air-dried for 5 minutes. This is followed by a treatment with protease reagent (Protease III)  Technologies, Cat# A-21206) for 2h at room temperature. Finally, the slides were dried and cover-slipped with Dako uorescence mounting medium (Cat# S302380-2, Agilent, CA), and scanned for analysis.

Data acquisition
All recordings were done at ve weeks after injection of the viral vectors. All sleep and respiration recordings were done in a plethysmography chamber (unrestrained whole-body plethysmograph, Buxco Research Systems) which allowed us to record the breathing of the mouse while continuously monitoring the gas in the chamber. Electroencephalogram (EEG) and electromyogram (EMG) were recorded using Pinnacle preamp cables connected to an analog adaptor (8242, Pinnacle Technology). Gas levels in the chamber were continuously monitored using CO2 and O2 monitors from CWE, Inc (Ardmore, PA, USA).
EEG, EMG, respiration, and CO2 and O2 levels were fed into an Axon Digidata 1322A analog-to-digital converter and the signals were acquired using Axoscope software-v10 (Molecular Devices, Foster City, CA, USA), or by acquired by the 1401 (CED, Cambridge, UK) and Spike2 ver.7 (CED, Cambridge, UK). Mice were connected to cables for sleep recording as well as with the ber-optic cables connected to the preimplanted glass ber in mice, for transmitting the laser light.
Mice were also placed in the plethysmography chamber beginning at 9:00 A.M. for 6 h during their lights-ON and behaviorally inactive period, on each test day for these recordings. Here, they underwent either the Laser-ON or Laser-OFF protocols, separated by a week and in random order.
During the Laser-ON protocol, either 473nm or 593nm laser was ON for 5, 10 0r 60s followed by 5 mins off. With 473nm, the photo-stimulations were done at either 5, 10 or 20Hz with pulse width of 10ms, and these stimulations were done for either 5 or 10s on different days, with a period of 6-7days between each treatment. During photo-activation using the 473nm laser, only normocapnia air was used in the chamber. The inhibitory 593nm laser stimulations were continuous for 60s, and preceded each 30s of CO2 stimulus by 20s and lasted 10s after the hypercapnia stimulus. In the Laser-OFF condition, everything was the same, except that the laser light was not turned on. The gas input for the plethysmograph was switched either to normocapnic air (21% O2, 79% N2) or hypercapnic air (10% CO2, 21% O2, and 69% N2) for 30 sec with 5 minutes in between the two hypercapnic stimuli. For both the photo-activation and inhibition experiments, trials were analyzed for latency to arousal and respiratory changes only for those epochs where the mouse was in NREM sleep for at least 30 s before the stimulus onset. We also used the splitter -TM105FS1B (Thorlabs, NJ) to split the laser stimulus for bilateral activation or inhibition. We adjusted the laser such that the light power exiting the bilateral beroptic cables was 8-10 mW, and this was checked before and after the experiment. The light power estimated at the PB is less than10 mW/mm 2 (www.stanford.edu/group/dlab/cgi-bin/graph/chart.php), and a similar range has been used by most researchers and by us earlier 19,55,62,63 . Note that this is probably a high estimate because some light is probably lost at the interface between the ber-optic cable and the implanted optic-ber.

Data analysis
Latency and respiratory data analysis: EEG arousals in response to CO2 were identi ed by EEG transition from NREM to a waking state, which was usually accompanied by EMG activation, as described previously. The latency of all the EEG arousals after onset of stimulation were scored and were compared across the Laser-ON and Laser-OFF days. Respiratory data was analyzed by running the respiratory script in the Spike2 (CED, UK) software, which performs breath by breath analysis for many respiratory parameters such as RR, V T and MV. For both latency and respiratory data, the ranges for analysis were selected by individuals that were blind to the treatment groups, who based the selection off the following Inscopix calcium-image processing: Calcium recording les were spatially ltered and motion corrected to correct the rigid brain movements using the Inscopix data processing (IDPS ver1.6). To extract the calcium activity traces from the individual cells, we used manually drawn small regions of interest. Raw traces were converted to ΔF/F (F-F baseline average / F baseline average), where F was the uorescent at any given point and F baseline average was the average baseline uorescence. These baseline image calculations were performed by the IDPS to derive ΔF/F value for each cell.
Statistical analysis: All statistical analyses were performed using SigmaPlot 12.3 (Systat Software, Inc.). For statistical comparisons, we rst con rmed if the data meets with the assumptions of the ANOVA, then either one way or two-way ANOVA was performed to compare the effects between various treatment groups. If differences in the mean values among the treatment groups were greater than would be expected by chance; then all pairwise multiple comparisons were performed using the Holm-Sidak method. The F and P values are described in the results section with details of the statistical tests also given in the respective gure legends and represented in the gures. The 'n' is reported in the gures and results and represents the number of animals, and the error bars represent mean± SEM. Using SigmaPlot 12.3, we also tested the sample size and power of the tests post hoc and found that the power of each statistical test was at least 80% at alpha= 0.05, suggesting adequate sample sizes for all the experiments. A probability of error of less than 0.05 was considered signi cant.

Declarations
Contribution of authors: SK: experimental design, data collection, analysis and manuscript writing; NL, JDL, RCT-data collection and analysis; YS-data analysis for the ber photometry and calcium imaging; SSB-maintaining the mouse breeding program and conducting insitu-hybridization; CBS-experimental design, data analysis and manuscript writing. The authors declare no competing interests. portions of the PB labeled immunohistochemically for both the immediate early gene cFos (green) and FoxP2 (red), from a mouse that was exposed to 2h of either normocapnic room air (columns a and b) or 10% CO2 (columns c and d). The insets in a and c demarcate the areas that are magni ed in the b and d.
Double-labeling (yellow nuclei) was prominent in the KF and in the central lateral FoxP2 clusters in mice exposed to CO2 but not those breathing normocapnic air.  In-vivo activity of individual PB FoxP2 neurons during CO2 exposure: Gradient-index (GRIN) lens were implanted above the injection sites in the PB of FoxP2-cre mice injected with Cre-dependent AAV-GCaMP6s (a) and the calcium activity pro les (ΔF/F) of individual PB Foxp2 neurons in response to the CO2 was acquired. Representative intracellular calcium activity of 3 neurons is shown for 30 sec before and 60 sec after onset of CO2 (b) and in the same trial for 10 sec before and 60 sec after onset of CO2 (c). Two cells (marked by green and yellow arrowheads whose activity pro les are also plotted in green and yellow) shown in b had uorescence that peaked at about 17-19 sec after exposure, before the maximal changes in respiration. A third cell (marked by a magenta arrow and activity pro le) peaked roughly 50 sec after onset of the CO2 trial (c), but while CO2 levels were still high. A representative trial that showed no cortical arousal during CO2 exposure but had a clear respiratory response to the 8% CO2 as seen by a gradual rise in the respiratory (RR) and the minute ventilation (MV) is shown in d. The ΔF/F from 9 cells is also plotted, showing the overall increase in intracellular calcium across the population, with most cells showing peaks at the time of maximal respiration, but with substantial variability across neurons (d). A heat map of the mean ΔF/F over 4-7 trials (during which animals exposed to 8% CO2 did not awaken) is shown for all 28 cells in e (blue to red-shows low to high ΔF/F). Note that although the RR and MV summated over these trials increased relatively smoothly (f), the activation of the PB FoxP2 neurons occurred in waves, and of the 17 cells that showed 3 peaks, 10 of them showed second and third peaks that were 17-19 sec apart and were synchronous in time after CO2 exposure, but that not every neuron participated in each wave of excitation.
Two way ANOVA compared the changes in RR and MV post CO2 to the Pre-Co2 baseline, where ***-P<0.001.

Figure 4
Effect of photoactivation of PB FoxP2 neurons on respiration: FoxP2-cre mice were injected bilaterally targeting the PB with cre-dependent AAV-Flex-ChR2-mCherry (red a, b; also immunostained for FoxP2 (green orescence, b2,3,5) and implanted for EEG/EMG recording and with bilateral glass optical bers to target illumination of the ChR2-expressing FoxP2 neurons (b1,2). The right side of the coronal section in b1, is also shown in b2 with immune-labeling for Foxp2 (green). The area within the box in b2 is shown at higher magni cation in b3-b5, and the doubly labeled neurons (yellow) are shown at higher magni cation in the inset in b5 (scale= 50 µm). Scale in b1-b5=100µm.
Respiration was analyzed for 5 breaths pre-stimulation, then for 5 breaths just before the end of stimulation or before cortical arousal and for 5 breaths after cortical arousal when stable breathing was attained without EMG artifacts. A representative trial with stimulation at 20 Hz (in normocapnic air) showed gradually increasing respiration which preceded the cortical arousal that occurred at 4.5 sec in this trial (c). In trials with 5 sec stimulation, the animals on an average woke up around 15 sec after the stimulation stopped (d), suggesting that the awakening was not due to the stimulation of the PB FoxP2 themselves, but may have been elicited by some aspect of the response to stimulation (e.g., respiratory efforts). Trials with stimulation for 10 sec usually caused EEG arousal either just before or after the termination of stimulation. Arousal latency was decreased dramatically (by 83%) by exposing the mice to continuous 2% CO2 during opto-stimulation (d, shown in yellow rectangle). A representative trial of stimulation at 20Hz for 10sec with continuous 2% CO2 is shown in e. Graphs in f compare the RR, V T and MV in mice subjected to laser stimulation at 5, 10 or 20Hz vs. no laser (Laser-OFF) for either 5 or 10s during normocapnia. Graphs in g compare the RR, V T and MV parameters in the mice subjected to laser stimulation at 20Hz vs. no laser (Laser-OFF) for 10s, in mice continuously exposed to 2% CO2 (as shown in e). The average values (mean ± SEM) are plotted for 5 breaths before onset of stimulation, during stimulation but before cortical arousal and in the post stimulation period after the cortical arousal when stable breathing is attained without EMG artifacts (e-g). Two-way ANOVA was used for statistical comparison, where **= P<0.01 (vs. pre-stimulation/ Laser OFF); ***= P<0.001 (vs. pre-stimulation/ Laser OFF), #= P<0.05 (vs. Laser-ON at normocapnia).

Figure 5
Descending projections of the PB FoxP2 neurons: Bilateral injections of Cre-dependent AAV-ChR2 (magenta) into the PB in a FoxP2-Cre mouse (a1) is shown with immuno-labeled for FoxP2 (green); nearly Supplementary Files